Protein hydrolysate and plant sterol containing composition for improving serum lipid profile and preventing atherosclerosis

ABSTRACT

The invention relates to a terapeutical composition comprising a protein hydrolysate and a plant sterol, wherein the weight ratio of the plant sterol to the protein hydrolysate is from 1:0.02 to 1:150. The invention also relates to a terapeutical composition comprising a protein hydrolysate and a synthetic emulsifier and/or a lipid based grain fraction having emulsifying properties. Said compositions can be used in a pharmaceutical, nutraceutical or food product for improving serum lipid profile.

FIELD OF INVENTION

The present invention relates to the field of nutrition and health.Especially it concerns improved compositions for oral use suitable forimproving serum lipid profile including reducing serum total and/or LDLcholesterol and/or apolipoprotein B levels.

BACKGROUND OF THE INVENTION

Cardiovascular disease (CVD) is one of the major causes of death inwestern world. Elevated serum total and/or LDL (low density lipoprotein)cholesterol and triacylglycerol levels, as well as low ratio of HDL(high density lipoprotein) cholesterol to LDL cholesterol, are some ofthe major risk factors for CVD. Recently also serum levels ofapolipoprotein B 100 (apo B) has been shown to be a reliable CVD riskmarker. In most developed countries a substantial amount of thepopulation have serum cholesterol levels not within the recommendedlevels. One of the first steps in improving the serum lipid profile ischanges in life style including changes in diet and exercise. It seems,however, difficult to change dietary habits and to follow dietaryrecommendations. Thus, there is a clear need for solutions beyondregular diet and lifestyle changes by which serum lipid profile can beimproved.

Food products enriched with components having cholesterol loweringeffect beyond normal nutrition have been commercially available for sometime. Representative examples are food products enriched with plantstanol or sterol fatty acid esters. Stanol fatty acid esters and thecholesterol lowering effects thereof are disclosed in U.S. Pat. No.5,502,045 as well as a suitable method for their preparation. Dietaryintake of 2 to 3 g/day of plant stanols is reported to lower serum LDLcholesterol levels in man up to 14% without affecting HDL cholesterollevels. The amount of atherogenic apolipoprotein B particles in blood isalso decreased, thus reducing the risk of CVD.

Some proteins have also been used to improve serum lipid profile.Proteins that have been shown to lower serum total and/or LDLcholesterol include e.g. soy protein (Anderson J. W., et al, New Engl.J. Medicine 1995 333 (5) 276-282), whey protein (Nagaoka et al., Agric.Biol. Chem. 1991 (55) 813-818) and wheat gluten (EP 0 790 060 A1). FDAhas approved a health claim about the role of soy protein in reducingthe risk of CVD by lowering blood cholesterol levels. In order toqualify for this health claim, a food must contain at least 6.25 gramsof soy protein per serving, the amount being one-fourth of the effectivelevel of 25 grams per day, and be included in a diet low in saturatedfat and cholesterol.

EP 0 669 835 B1 describes a hypocholesterolemic dietary compositioncontaining soy protein and sitosterols, the sitosterols and soy proteinbeing of natural origin and having synergistic cholesterol loweringeffect. The composition is used for example as tablets, capsules orsyrups. Effective daily intakes of the composition are not given Also EP1 046 396 A2 describes a composition containing soy protein, preferablysoy protein isolate or soy protein concentrate, and plant sterols havingsynergistic cholesterol lowering effect. The synergistic effect isachieved, when both the soy protein and plant sterols are administeredin amounts that would be sufficient to induce cholesterol-loweringactivity of each of the administered compound alone. WO 01/37681describes a composition containing phytosterols and an isolatedwater-soluble protein, such as soy protein or caseinate, and optionallyalso an emulsifier. The composition can be utilized in food products,where the composition improves mouthfeel and stability of the sterolcontaining foods. No improved cholesterol lowering effect compared tothe effect achieved with phytosterols alone is attributed to thiscomposition.

U.S. Pat. No. 6,113,972 describes a phytosterol protein complex, wherethe complex increases the bioavailability of the phytosterols. Nocholesterol lowering effect is attributed to the protein part of thiscomposition, but the protein functions only as a carrier to thephytosterols. In fact, any fat binding protein can be used in thiscomplex and especially egg proteins are preferred. A compositioncontaining plant sterols/stanols and an emulsifier, mixed into aprotein-containing product, is described in WO 00/33669. The proteindoes not have any cholesterol lowering effect in this compositioneither, but it aids in the formation of the emulsion.

Protein hydrolysates have been shown to have greater serum cholesterollowering effect than the corresponding unhydrolyzed proteins (Sugano etal., J. Nutr. 1990 (120) 977-985). The cholesterol lowering effect ofprotein hydrolysates might be attributed to certain peptides, forexample such as found from bovine milk β-lactoglobulin by Nagaoka et al.(Biochem. Biophys. Res. Commun., 2001, 281(1), 11-17), but this stillremains to be proven by well-controlled clinical and/or animal studies.EP 0 790 060 A1 discloses a protein hydrolysate/phospholipid complex forimproving lipid metabolism, where the complex contains 10% or more boundphospholipids, especially lecithin or enzyme modified lecithin. Lipidprofile improving effect, especially LDL-cholesterol lowering andHDL-cholesterol raising effect of lecithin is well known in prior art(for example Childs et al., 1981, Atherosclerosis 38, 217). In thecomplex according to EP0790060 A1 certain bound phospholipids (lecithinor enzyme modified lecithin) are believed to remarkably contribute tothe lipid metabolism improving effect of the complex.

The current literature and especially FDA's interim approval of a healthclaim for plant sterols have increased the interest of the food industryin supplementing foods with plant sterols. Indeed, many such food itemshave recently been introduced into the market. It is assumed that newplant sterol containing foods will appear into the market rapidly.

The active launching of new plant sterol containing foods has raised aconcern that a part of the population may have higher daily intake ofplant sterols than what is needed to obtain an optimal cholesterollowering effect. Short-term high intake of plant sterols has not beenshown to be harmful. However, there is no data on possible long-termside effects of ingestion of high daily amounts (>3 g/day or higher) ofplant sterols. Furthermore, there is currently not a clear understandingof the biological impact of the increased serum level and thus highersystemic availability of plant sterols caused by increased intake fromsterol enriched foods, especially foods enriched with unsaturated plantsterols.

There is a clear need for improved solutions by which the serum lipidprofile can be improved, e.g. elevated serum total and/or LDLcholesterol levels reduced and/or HDL cholesterol levels increasedand/or triacylglycerol levels reduced and/or apolipoprotein B levelsreduced. Compositions based on combinations of active ingredients havingenhanced, additive or even synergistic effects compared to the activeingredients alone are needed. It would also be desirable to obtainmeaningful and sustainable improvements in the serum lipid profile withlower daily intake of the individual active ingredients than what isobtainable with the intakes currently used. Possible adverse effects ofthe active ingredients such as possible impact on fat-solubleantioxidants and other digestive discomforts could thus be reduced.Incorporation of the active ingredients into a wider range of foodproducts without adverse effects on organoleptic properties is alsoneeded. The present invention provides compositions meeting theserequirements.

SUMMARY OF THE INVENTION

The present invention provides improved compositions for improving serumlipid profile in humans and/or animals and a method for improving serumlipid profile in humans and/or animals by orally administering thecomposition. In one aspect the compositions comprise combinations ofprotein hydrolysates and plant sterols. The compositions may optionallycomprise also emulsifier(s) and/or fat and/or mineral salt. Thecompositions comprise one or more protein hydrolysates in combinationwith one or more plant sterols (sterols and/or stanols) in their freeand/or esterified forms, optionally in combination with one or moreemulsifiers and/or fat and/or mineral salt. Preferably the emulsifier isused to produce a complex with protein hydrolysates and/or sterols. In asecond aspect the compositions comprise combinations of proteinhydrolysates and synthetic emulsifier(s). The compositions mayoptionally comprise also fat and/or mineral salt. In a third aspect ofthe invention the compositions comprise combinations of proteinhydrolysates and lipid based grain fractions having emulsifyingproperties. The compositions may optionally comprise also fat and/ormineral salt.

The compositions can be used as such or as pharmaceuticals ornutraceuticals or most advantageously in food products. Typically suchan improved composition is a complex containing soy protein hydrolysate,emulsifier and plant sterols, advantageously used in food products forimproving the serum lipid profile.

It was found that the combination of protein hydrolysates and plantsterols, optionally with emulsifiers and/or fat and/or mineral salt,further enhance the serum lipid profile improving effect that isobtained by using the individual active ingredients and even worksynergistically in improving the overall serum total lipid profile, forinstance in reducing blood serum total and/or LDL cholesterol levels,and have greater effect than the additive effect that was expected. Inaddition some of the combinations, especially those containing soyprotein hydrolysate showed beneficial effect on HDL cholesterol levels.Also the lowering of serum triglyceride levels was shown to besurprising effective.

The present invention provides also means for further improving of serumlipid profile by using combinations of the active ingredients comparedto the effects obtained when one of the ingredients is used alone. Forexample using the compositions of the present invention in combinationwith a healthy recommended diet provides means by which serum LDLcholesterol level can be reduced even by 25% or more. The presentinvention thus provides a versatile non-drug approach to improve serumlipid profile, e.g. to lower serum total and/or LDL cholesterol,especially in subjects having serum lipid levels not within therecommended limits. The present invention also provides combinations ofthe active ingredients, especially those containing mineral salt and/orspecific protein hydrolysates, that have beneficial effect on bloodpressure. The combination of the active ingredients according to thepresent invention thus reduces the risk of CVD beyond what is obtainableby using only one of the active ingredients.

The present invention further provides means for minimizing the dailyintake of plant sterols in humans and still achieving a similar serumlipid profile improving effect as obtained with recommended daily intakeof plant sterols from commercial plant sterol enriched products. This isespecially important as the daily amount of plant sterols needed forobtaining a reduction in serum total and/or LDL cholesterol can bereduced, thus preventing potential over-consumption of plant sterols.Furthermore, the commercial availability of plant sterols is limited. Inaddition the commercial crude plant sterols are expensive due to theirlimited availability, whereas the availability of raw materials forprotein hydrolysates or protein hydrolysate complexes usable accordingto this invention is not restricted.

By the use of the compositions according to the present invention theplant sterol supply can be more effectively utilized with the aim ofreducing the risk of CVD in subjects as well as in the whole population.The present invention provides a way of obtaining an enhanced serumlipid profile improvement or a similar lipid profile improvement asobtained with plant sterols or protein hydrolysate based ingredientsalone, but especially with lower daily intake of plant sterols and/orprotein hydrolysate. As active ingredients based on protein hydrolysatesare cheaper than plant sterols the present invention also provides a wayof obtaining an optimal lipid profile improving effect more costeffectively.

Compared to combinations of plant sterols and unhydrolyzed proteinspreviously known in the art, the compositions of the present inventioncomprising the combination of plant sterols and protein hydrolysates,optionally with emulsifiers and/or fat and/or mineral salt, haveenhanced serum lipid profile improving effect and can also be utilizedin a wider range of food products and by a wider range of consumers. Inaddition to having greater cholesterol lowering effect compared tounhydrolyzed proteins, protein hydrolysates are also less allergenicthan unhydrolyzed proteins, thus enabling wider range of consumers beingable to benefit the serum lipid profile improving effect. Thecompositions of the present invention can also be more easilyincorporated into food products, such as beverages, without adverseeffects on the texture due to the reduced viscosity of some of theprotein hydrolysates compared to unhydrolyzed proteins.

A problem encountered with protein hydrolysates and their incorporationinto food products is often a pronounced bitter taste of thehydrolysate. The bitter taste is the result of cleavage of the proteinsat amino acids with hydrophobic side chains, which results in formationof peptides with exposed hydrophobic amino acid residues. Hydrolysatesmay thus have an adverse effect on the taste of the food product wherethey are incorporated. In addition to protein hydrolysates, also someprotein hydrolysate/phospholipid complexes have bitter taste and canadversely affect the taste of the products containing them. In additionto off-flavors, some protein hydrolysate/phospholipid complexes,especially complexes containing soy protein hydrolysate and lecithin ormodified lecithin, have also strong yellowish or brown colour and theiruse in light coloured products, such as some dairy products, may thuscause off-colours to the product.

The present invention by combining plant sterols and proteinhydrolysates, optionally with emulsifiers and/or fat and/or mineralsalt, in lipid profile improving products, such as foods, provides a wayof obtaining at least similar lipid profile improving effect, especiallycholesterol lowering effect, as can be obtained with proteinhydrolysates alone, but with lower protein hydrolysate content and thuswith less adverse effect on the taste of the food products. Complexingprotein hydrolysates either with synthetic emulsifiers or with plantsterols or advantageously with both emulsifiers and plant sterols alsodiminishes the unpleasant taste of protein hydrolysates. Thecompositions according to the present invention thus widen the range offood products in which the protein hydrolysates can be incorporated toinclude also mild-tasting ones and still achieving at least similarserum lipid profile improving effect. The compositions of the presentinvention also widen the range of food products in which proteinhydrolysate/emulsifier complexes can be incorporated to include alsolight coloured products. The present invention thus also provides amethod to improve the sensory properties of lipid profile improvingfoods containing protein hydrolysates or protein hydrolysate/emulsifiercomplexes.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the present invention there is provided a compositioncomprising a protein hydrolysate and a plant sterol, wherein the weightratio of the plant sterol to the protein hydrolysate is from 1:0.02 to1:150.

The composition of the present invention can additionally comprise anemulsifier and/or a fat. Furthermore, the composition can comprise amineral salt.

The weight ratio of the plant sterol to protein hydrolysate in thecompositions is preferably from 1:0.2 to 1:30, more preferably from1:0.4 to 1:12.5 and most preferably from 1:1 to 1:5.

As used here, the term “protein hydrolysate” includes all hydrolyzedproducts of proteins prepared by using a proteolytic enzyme preparation,a microorganism containing suitable proteolytic activity or acidhydrolysis or any combination thereof, and having serum lipid profileimproving effect. Commercially available hydrolysates can be used orhydrolysates can be prepared. Preferred hydrolysates have a molecularweight of 300-100 000 D, more preferably 500-50 000 D, most preferably500-30 000 D and/or they are only slightly soluble in water.

Plant, animal or microbial proteins and/or their mixtures can be used asprotein sources for the hydrolysates. Proteins of vegetable origin arepreferred. Most preferred are proteins of grain or legume origin.Suitable vegetable protein sources are for example soybean protein,wheat protein, especially wheat gluten, corn protein, oat protein, ryeprotein, rapeseed or canola protein, barley protein, rice protein,flaxseed protein, potato protein, pea protein, lupin protein, maizeprotein and buckwheat protein. Of animal origin, suitable examplesinclude milk proteins, such as whey protein, and their fractions. Allthese proteins can be used in different commercially available purifiedor non-purified forms as source for the hydrolysates. Also materialscontaining these proteins and other major constituents, such ascarbohydrates, can be used as source for the hydrolysates.

Protein hydrolysates are preferably prepared via enzymatic hydrolysis bymethods and processes well known in the art, but can also be preparedusing other known hydrolytic techniques, such as acid hydrolysis. Forexample EP 0 790 060 A1 discloses a method for preparing suitableprotein hydrolysates. Typical preparation methods include treatment ofthe protein moiety with a proteolytic enzyme in aqueous medium, wherethe protein or protein containing material is dispersed in water and pHis adjusted with an acid or a base to the optimum pH range of the enzymeto be used. The enzyme is preferably added in an amount of 0.2-5%, morepreferably 0.5-2% based on the substrate protein and the reaction iscarrier out at optimum temperature and pH for the time needed dependingon the enzyme and the degree of hydrolysis wanted. The reaction istypically terminated by heating the mixture to a temperature high enoughto inactivate the enzyme. The reaction mixture can optionally beneutralized before or after the heating step by using a suitable acid,e.g. hydrochloric acid, or a base, e.g. sodium hydroxide. Preferably thefraction having serum lipid profile improving effect is then separatedfrom the reaction mixture. In case the water insoluble or slightly watersoluble fraction of the reaction mixture is desired, it can be separatede.g. by centrifugation or filtration techniques to obtain what is herereferred to as a protein hydrolysate slurry. The protein hydrolysateslurry can be used as such or as washed with water and/or dried. Theprocess used for drying is not critical, for example freeze-drying,spray drying or any other process known in the art may be used, whichproduces a powder either directly or through a grinding step. Dried andpowdery protein hydrolysate is especially preferred as it entraps lipidsefficiently. Proteolytic enzymes that can be used in the preparation ofthe hydrolysates of the present invention include enzymes from plant,microbial and animal origin, including also enzymes from geneticallymodified organisms, that at suitable reaction conditions hydrolyseproteins to hydrolysates that have a serum lipid profile improvingeffect. Suitable proteolytic enzymes are e.g. pepsin, trypsin,pancreatin and papain. It is also possible to use different enzymessequentially, e.g. pepsin and trypsin or pepsin and pancreatin.Especially preferred are the enzymes or combinations of enzymes thatproduce protein hydrolysates that are only poorly digestible or nolonger digestible in human intestine and can thus be referred also as“resistant proteins”.

As used here, the term “plant sterol” refers to any sterol as defined inthe following and having lipid profile improving effect. Plant sterolsinclude both sterols and saturated sterols i.e. stanols in either theirfree form or as esterified e.g. with fatty acids (2-24 carbon atoms,saturated, monounsaturated or polyunsaturated, including also specialfatty acids such as conjugated fatty acids, e.g. CLA, and EPA and DHA),hydroxybenzoic acids or hydroxycinnamic acids (ferrulic or coumaricacids) or other organic acids such as e.g. di- or tricarboxylic acidsand/or hydroxy acids or with any combination of the said acids. Inaddition, the definition of plant sterols also include glycosidicsterols and their derivatives. Any combinations of the free and variousesterified and glycosylated forms are also included.

As used here, the term “plant sterol in esterified form” or “plantsterol ester” refers to plant sterols having at least 60%, preferably atleast 85%, most preferably at least 95% of the plant sterols inesterified form.

In this specification the sterols include 4-desmethyl sterols,4-monomethyl sterols and 4,4-dimethyl sterols (including triterpenealcohols) and the stanols include 4-desmethyl stanols, 4-monomethylstanols and 4,4-dimethyl stanols. Typical 4-desmethyl sterols aresitosterol, campesterol, stigmasterol, brassicasterol,22-dehydrobrassicasterol, Δ5-avenasterol. Typical 4,4-dimethyl sterolsare cycloartenol, 24-methylenecycloartenol and cyclobranol. Typicalstanols are sitostanol, campestanol and their 24-epimers, cycloartanoland saturated forms obtained by saturation of e.g. triterpene alcohols(cycloartenol, 24-methylenecycloartenol and cyclobranol). The definition“plant sterol” includes all possible natural blends or any mixtures ofnamed sterols and/or stanols as well as any individual sterol or stanol.

The amounts of plant sterols in this specification are calculated asplant sterol equivalents i.e. as the amount of free plant sterol.

Commercially available plant sterols in their free or esterified formcan be used as such. When plant sterols in their free form are used, theparticle size of the plant sterols is preferably reduced to enhance thedispersability, dissolvability and solubility of the plant sterols.Particle size reduction can be done by many techniques known in the art,e.g. by different dry or wet grinding or micromilling techniquesdescribed for example in U.S. Pat. No. 6,129,944, WO 98/58554 and EP 1142 494 A1. Other components, such as a suitable admixture can bepulverized together with plant sterols, the choice of the othercomponents depending on the food material, nutraceutical orpharmaceutical in which the active ingredients are to be added. Examplesof the admixture include various structure and flavor enhancers, as wellas flours especially in case the active ingredients are to be added intobakery products.

Plant sterols in their free form may also be used molten, especially incompositions containing an emulsifier and/or a fat. Preferably ahomogenous mixture that is easily used in the compositions of thepresent invention is formed from plant sterols and an emulsifier and/ora fat by heating plant sterols to their melting point, to 60-150° C.,typically to 130-150° C., and adding the emulsifier and/or fat to thesterols, either prior to or after heating. Suitable techniques that canbe utilized are described e.g. in U.S. Pat. No. 6,190,720 B1. Mostpreferably a blend of plant sterols and emulsifier(s) and/or fat isheated until the components are dissolved. The mixture is cooled underagitation prior to adding it into the compositions of the presentinvention.

Preferably the plant sterol is esterified and most preferably it is aplant sterol fatty acid ester. The fatty acid ester is technically verysuitable for incorporation into a wide range of different products andis especially preferred as it has very good organoleptic properties,enabling production of the compositions of the present invention withgood organoleptic properties.

Preferably the plant sterol is a stanol because its absorption isnegligible and the use of stanol is therefore safer. Also thecholesterol lowering effect seems to be stronger with stanols. Mostpreferred are therefore the stanol fatty acid esters for use in thecompositions according to the invention.

As used here, the term “emulsifier” refers to a substance promoting theformation and improving the stability of emulsions. Emulsifiers aresynthetically produced, derived through chemical modification ofnaturally occurring materials or natural products. The characterizingfeature of emulsifiers is a structure in which one portion of themolecule is polar (hydrophilic) and the other non-polar (hydrophobic),which allows the emulsifier to align and stabilize the contact surfacesof the two immiscible phases of the emulsion. The relative size andcharacter of the polar and non-polar portions of the molecule, amongother factors, affect the type and quality of emulsions produced. HLB(hydrophilic lipophilic balance) scale is commonly used to generallydescribe the emulsifying properties of emulsifiers. An emulsifier with alow HLB value (up to about 6) tends to promote W/O emulsions, anemulsifier with an intermediate HLB value W/O or O/W emulsions and anemulsifier with a high HLB value (from about 8) O/W emulsions.

Preferably the emulsifier or a mixture of emulsifiers is used to producea complex with plant sterols and/or protein hydrolysates. Typical, butnon-restricting examples of suitable emulsifiers include monoglycerides,such as distilled monoglycerides, diglycerides, monoglyceridederivatives, such as acetic, lactic, succinic or citric acid esters ofmonoglycerides, lecithins, modified lecithins such as enzyme modifiedlecithin, for example lysolecithin, polyglycerol esters, polyglycerolpolyricinoleate, sorbitan esters, polysorbates, propylene glycol esters,stearoyl lactylates, such as sodium stearoyl lactylate and calciumstearoyl lactylate, diacetyl tartaric acid esters, diacetyl lactic acidesters, sugar esters, and mixtures thereof. Also lipid based grainfractions having emulsifying properties can be used as emulsifiers.These include lipid fractions e.g. from oat.

Preferred emulsifiers or mixtures of emulsifiers are those having a HLBvalue or emulsifying properties equivalent to a HLB value above 4, morepreferably above 6. Examples of preferred emulsifiers include acetic,succinic, lactic and citric acid esters of monoglycerides, lecithins,modified lecithins, polyglycerol esters, polysorbates, diacetyl tartaricacid esters, monoglycerides, stearoyl lactylates and mixtures thereof.

As used here, by “synthetic emulsifier” is meant emulsifiers that aresynthetically produced or derived through chemical modification ofnaturally occurring materials. These synthetic emulsifiers do not havestrong color, unlike some naturally occurring emulsifiers, such aslecithin and enzymatically modified lecithin. The synthetic emulsifierscan thus be used as a part of protein hydrolysate/emulsifier complexesadded also into light colored products. The synthetic emulsifier arealso often more pure than the naturally occuring emulsifiers. Suitable,but non-limiting examples of the synthetic emulsifiers are monoglyceridederivatives, polyglycerol esters, propylene glycol esters, polysorbates,sorbitan esters, stearoyl lactulates, diacetyl tartaric acid esters,sugar esters and mixtures thereof. Preferred synthetic emulsifiers ormixtures of synthetic emulsifiers are those having a HLB value oremulsifying properties equivalent to a HLB value above 4, morepreferably above 6.

As used here, the term “complex” is a composition in which only a partof the emulsifier(s) and/or fat and/or plant sterols are extractable byusing non-polar solvents commonly used to extract “free lipids”. Thesenon-polar solvents include e.g. petroleum ether and hexane. The rest ofthe emulsifier(s) and/or fats and/or plant sterols can only be extractedafter hydrolysis e.g. by using methods including acid hydrolysis, suchas AOAC 922.06.

As used here, by “fat” is meant edible solid fats, semi-solid fats,liquid oils and any mixtures thereof. Also fat substitutes such as sugarpolyesters are included. Examples of solid and semi-solid fats that areusable include tallow, lard, butter, margarine and shortenings as wellas semi-solid tropical oils, such as coconut oil and palm kernel oil.Typical examples of liquid oils include canola/rapeseed oil, soybeanoil, sunflower oil, olive oil, palm oil, corn oil, sesameseed oil,cottonseed oil, wheat germ oil, peanut oil and fish oils. Liquidvegetable oils are especially preferred. The fat can be naturallyoccurring or modified, for example hydrogenated, transesterified,contain structured triacylglycerols or an increased amount ofdiacylglycerols.

As used here, the term “mineral salt” includes nutritionally beneficialmineral salts having elevated K and/or Mg and/or Ca content and/orreduced Na content compared to common salt. In U.S. Pat. No. 6,136,349this kind of mineral salt, especially when administered together withplant sterols and/or used to replace common salt in food items, issuggested to bring beneficial health effects, such as lowering elevatedblood pressure. Examples of suitable mineral salt compositions includemineral salt sold by trade name Pansalt® or Cardia® salt. The mineralsalt is preferably added into the protein hydrolysate, more preferablyinto the protein hydrolysate slurry or washed protein hydrolysate.

In the following some typical compositions of the present invention andthe preparation thereof will be explained in more detail.

The composition of the present invention comprising one or more proteinhydrolysates and one or more plant sterols can be used as such or as apharmaceutical or a nutraceutical or most advantageously in foodproducts for improving serum lipid profile in man. The proteinhydrolysate(s) and plant sterol(s) may be added in any suitable way intofood products, pharmaceuticals or nutraceuticals separately asingredients or they may be suitably combined to form compositions orcomplexes before forming the composition according to the invention.Preparation of the compositions and the incorporation into foodproducts, pharmaceuticals or nutraceuticals can be facilitated byreducing particle size of said compositions or components thereof e.g.by grinding or micromilling. Also other components such as water ormineral salt may be added. Typical but non-restricting examples of thepreparation of the compositions and complexes are described in thefollowing embodiments.

In one embodiment of the present invention the protein hydrolysate(s)and the plant sterol(s) are added into food products, pharmaceuticals ornutraceuticals separately as ingredients. The active ingredients areadded into food material, pharmaceuticals or nutraceuticals byconventional processes for producing these products.

Commercially available plant sterols in their free form can be used assuch, or more preferably the particle size of the plant sterols isreduced to enhance the dispersability and solubility of plant sterols asdescribed above. Molten plant sterols in their free form can also beused. Advantageously the main part of plant sterols is in esterifiedform and preferably melted, if needed, before addition into foodproducts, pharmaceuticals or nutraceuticals. Protein hydrolysates can beused in any suitable form including protein hydrolysate slurry, washedand/or dried protein hydrolysate, protein hydrolysate in an aqueoussolution or protein hydrolysate with reduced particle size.

In another embodiment of the present invention the proteinhydrolysate(s) and the plant sterol(s) are combined into a compositionthat can be used as such or added into pharmaceuticals or nutraceuticalsor food products. When plant sterols in their free form are used, theyare mixed with dried protein hydrolysates to form a homogenouscomposition or more preferably homogenized with the protein hydrolysateslurry or water washed protein hydrolysate to form a proteinhydrolysate/plant sterol complex. The mixture may be used as such orpreferably be dried by methods commonly used in the art, e.g. by freezedrying or spray drying, to form powder either directly or through agrinding step. Commercially available plant sterols can be used as suchor preferably with reduced particle size. In a preferred embodiment themain part of the plant sterols is in esterified form. Esterified plantsterols are preferably mixed with a dried and powdery proteinhydrolysate to form a protein hydrolysate/plant sterol complex.

In still another embodiment of the present invention the proteinhydrolysate(s), mineral salt and the plant sterol(s) are combined into acomposition that can be used as such or added into pharmaceuticals ornutraceuticals or food products. The mineral salt is preferably addedinto the protein hydrolysate, more preferably into the proteinhydrolysate slurry or washed protein hydrolysate and the mixture iscombined with plant sterols.

Alternatively the compositions comprising the protein hydrolysate(s) andthe plant sterol(s) can be prepared by exposing a mixture ofunhydrolyzed protein(s) and plant sterol(s) to hydrolysis, preferably toenzymatic hydrolysis by methods and processes known in the art.

The composition of the present invention comprising one or more proteinhydrolysates, one or more emulsifiers and/or fats and one or more plantsterols can be used as such or as pharmaceuticals or nutraceuticals ormost advantageously in food products for improving serum lipid profilein man. The protein hydrolysate(s), plant sterol(s) and emulsifier(s)and/or fat(s) may be added in any suitable way into food products,pharmaceuticals or nutraceuticals separately as ingredients or they maybe suitably combined to form compositions or complexes before formingthe composition according to the invention, which is incorporated in thefood products, pharmaceuticals or nutraceuticals. Preparation of thecompositions and the incorporation into food products, pharmaceuticalsor nutraceuticals can be facilitated by reducing particle size of saidcompositions or components thereof e.g. by grinding or micromilling.Also other components such as water or mineral salt may be added.Typical but non-restricting examples of the preparation of thecompositions and complexes are described in the following embodiments.Preferably the emulsifier is used to produce a complex with proteinhydrolysate(s) and/or plant sterol(s).

In one embodiment of the present invention the protein hydrolysate(s)and the emulsifier(s) are used to form a protein hydrolysate/emulsifiercomplex, and the complex and the plant sterols are added into foodproducts, pharmaceuticals or nutraceuticals separately as ingredients.The active ingredients are added into food material, pharmaceuticals ornutraceuticals by conventional processes for producing these products.

The protein hydrolysate/emulsifier complex can be prepared by mixing theemulsifier and protein hydrolysate e.g. by a high speed mixer, or morepreferably by forming a protein hydrolysate/emulsifier complex by mixingthe ingredients in the presence of water. Preferably the emulsifier isdispersed in water, protein hydrolysate added, followed by thoroughmixing at room temperature. Optionally the complex is dried byconventional drying techniques, such as e.g. freeze-drying orspray-drying, to form a powdery product either directly or through agrinding step. The complex can also be prepared of the proteinhydrolysate and a mixture of emulsifier and fat, with or without thepresence of water. The protein hydrolysate/emulsifier complex containspreferably at least 5%, more preferably at least 10%, and mostpreferably at least 20% emulsifier on dry weight basis Also anycommercially available protein hydrolysate/phospholipid complex can beused as protein hydrolysate/emulsifier complex.

Prior art discloses protein/phospholipid and proteinhydrolysate/phospholipid complexes containing 10% or more boundphospholipid (EP 0 790 060 A1), especially lecithin or enzyme modifiedlecithin, where both the protein or protein hydrolysate andphospholipids have lipid metabolism improving effect. In the presentinvention a wider range of emulsifiers or their combinations can beused. This is of particular benefit when the complex is added into foodproducts, pharmaceuticals or nutraceuticals, where the emulsifiers canbe selected to bring advantageous effects on the properties, e.g.texture or taste, of the final product and not just solely on the basisof enhancing the lipid profile improving effect of the proteinhydrolysate.

Commercially available plant sterols in their free form can be used assuch, or more preferably the particle size of the plant sterols isreduced to enhance the dispersability and solubility of plant sterols.Particle size reduction can be done by many techniques known in the art,e.g. by grinding. Advantageously the main part of plant sterols is inesterified form and preferably melted, if needed, before addition intofood products, pharmaceuticals or nutraceuticals.

In another embodiment of the present invention plant sterols in a formof a plant sterol/emulsifier complex, or plant sterols dissolved orsuspended in a fat, and protein hydrolysate are added into foodproducts, pharmaceuticals or nutraceuticals separately as ingredients.The active ingredients are added into food material, pharmaceuticals ornutraceuticals by conventional processes for producing these products.The plant sterol/emulsifier complex or the suspension or solutioncontaining plant sterols and fat is prepared by dissolving or suspendingplant sterols in their free form, preferably with reduced particle size,in a melt of an emulsifier, a fat or a mixture of emulsifier and fat atelevated temperatures, preferably at >60° C., more preferably at >80°C., mixing to form a homogenous dispersion or solution and cooling withagitation. Also molten plant sterols may be used. The weight ratio ofplant sterols to emulsifier can be from 1:0.01 to 1:5, preferably from1:0.05 to 1:2, more preferably from 1:0.1 to 1:2. The crude fat content,excluding plant sterols and emulsifiers, is preferably less than 80%,more preferably less than 60%, most preferably less than 20% of theweight of the complex or dispersion or solution. Also plant sterolsmainly in their esterified form may be used instead of plant sterols intheir free form. Protein hydrolysates can be used in any suitable formincluding protein hydrolysate slurry, washed and/or dried proteinhydrolysate, protein hydrolysate in an aqueous solution, proteinhydrolysate with reduced particle size and proteinhydrolysate/emulsifier complex.

In a preferred embodiment of the present invention the plant sterol(s),the emulsifier(s) and the protein hydrolysate(s) are combined into aprotein hydrolysate/plant sterol/emulsifier complex that can be used assuch or added into pharmaceuticals or nutraceuticals or food products.

The complex is preferably prepared by first dissolving or suspendingplant sterols in a melt of an emulsifier, a fat or a mixture ofemulsifier and fat at elevated temperatures, preferably at >60° C., morepreferably at >80° C., mixed to form a homogenous dispersion or solutionand cooled with agitation. The weight ratio of plant sterols toemulsifier can be from 1:0.01 to 1:5, preferably from 1:0.05 to 1:2,more preferably from 1:0.1 to 1:2. The fat content, excluding plantsterols and emulsifiers, is preferably 0-80%, more preferably 0-60%,most preferably 0-20% of the weight of the complex or suspension orsolution. Protein hydrolysate in a dry form, as a slurry or a waterwashed protein hydrolysate or an aqueous protein hydrolysate suspensionor solution is then mixed and homogenized with the cooled plantsterol/emulsifier or plant sterol/fat or plant sterol/emulsifier/fatmixture to form a protein hydrolysate/plant sterol/emulsifier complex orif no emulsifier is present, a protein hydrolysate/plant sterol complex.The homogenized mixture can optionally be dried and/or its particle sizereduced before usage. Plant sterols in their free form are preferablyused. Plant sterols in their free form with reduced particle size areespecially preferred. Also molten plant sterols may be used. Plantsterols mainly in their esterified form may also be used. The complexmay also be prepared by combining a protein hydrolysate/emulsifiercomplex and plant sterols.

Alternatively the compositions consisting the protein hydrolysate(s),the emulsifier(s) and/or fat(s) and the plant sterol(s) can be preparedby first exposing a complex of unhydrolyzed protein/emulsifier orunhydrolyzed protein/plant sterols or unhydrolyzed protein/plantsterols/emulsifier(s) to hydrolysis, preferably to enzymatic hydrolysisby methods and processes known in the art and described earlier, andthen combining the hydrolyzed complex with other components according tothe present invention.

In a second aspect of the present invention there is provided acomposition comprising a protein hydrolysate and emulsifier(s) oremulsifier-fat mixture for improving serum lipid profile in man, whereinthe synthetic emulsifier(s) are used. By using synthetic emulsifiers,the taste, colour and impurity problems commonly attributed tocompositions containing natural emulsifiers can be avoided. Also thelipid based grain fraction(s) having emulsifying properties can be usedas emulsifiers in a composition comprising a protein hydrolysate andemulsifier(s) or emulsifier-fat mixture. In this third aspect of theinvention the protein hydrolysate and the emulsifier are perferablyobtained from the same source. E.g. oat is fractionated and a proteinrich fraction is hydrolysed and combined with a lipid rich fraction to amixture or a complex. A benefit with this combination is that it is madeof non-allergenic materials.

The composition of the present invention comprising one or more proteinhydrolysates and one or more synthetic emulsifiers and optionallyfat(s), and/or one or more lipid based grain fractions havingemulsifying properties, can be used as such or as pharmaceuticals ornutraceuticals or most advantageously in food products for improvingserum lipid profile in man. The protein hydrolysate(s) and syntheticemulsifier(s), and optionally fat(s), or the protein hydrolysate(s) andlipid based grain fractions having emulsifying properties may be addedin any suitable way into food products, pharmaceuticals ornutraceuticals separately as ingredients or they may be suitablycombined to form compositions or complexes before forming thecomposition according to the invention, which is incorporated in thefood products, pharmaceuticals or nutraceuticals. Preferably thesynthetic emulsifier or lipid based grain fraction is used to produce acomplex with protein hydrolysate(s) by methods described earlier.

In a further aspect of the present invention there is provided a foodproduct comprising at least one basic nutritional ingredient and atleast one of the compositions of the present invention as defined above.

The plant sterol constituent of the composition is present in a higheramount than naturally occurring in the basic nutritional ingredient(s).

The food product of the present invention can be prepared by adding saidcomposition to food material(s) by the conventional processes forproducing food products. The composition can be added as such (either inliquid, semi-solid or dried form) or the constituents of the compositioncan be added separately as ingredients.

The food product of the present invention can be in the form of variousfood compositions, including but not restricted to

-   -   bakery products and confectionery (fresh and dry bakery        products, e.g. fresh bread, other bread products, cakes,        muffins, waffles, biscuits, crackers, protein enriched bakery        products etc.)    -   cereal products and snacks (breakfast cereals, muesli, bars,        such as cereal based and muesli bars, such bars possibly        containing chocolate, pasta products, flours etc.)    -   beverages (alcoholic and non-alcoholic drinks, including e.g.        soft drinks, juices and juice-type mixed drinks, fortified        beverages such as e.g. protein or calcium fortified beverages,        probiotic drinks, sports and energy drinks, dietary supplement        and meal replacement drinks, concentrates or premixes for        beverages and powdered drinks where the content of compositions        of the present invention is calculated for the ready-to-use        form, etc.)    -   dairy products (milk, milk based products, e.g. cheese, cream        cheese and the like, yogurt, frozen yogurt, other frozen dairy        foods, drinkable yogurt, other fermented milk products, dairy        beverages, ice cream, desserts, spreads etc.)    -   fermented cereal products    -   sauces, soups    -   meat, fish, poultry and egg products (e.g. sausages, meat balls        etc.)    -   analogues for e.g. dairy or meat products (e.g. imitations of        cheese, yogurt, ice cream, desserts, meat substitutes, milk        alternatives etc.), non-dairy frozen desserts    -   soy based products    -   vegetable oil based products (e.g. margarines, spreads,        dressings, mayonnaise etc.)    -   ready mixes (for baking e.g. breads, cakes, muffins, waffles,        pizzas, pancakes; or for cooking e.g. soups, sauces, desserts,        puddings) to be used in preparing or manufacturing foods.

The food product of the present invention can also contain othernutritionally beneficial components, some of which may further enhancethe effects of the compositions of the present invention. The food canbe fortified with these components or the components can be an intrinsicpart of the other food ingredients.

Examples of the nutritionally beneficial components includediacylglycerol and n-3 fatty acids, e.g. from fish oil, which haveadvantageous effects on the lipid metabolism and may thus reduce therisk of cardiovascular disease. N-3 fatty acids act favourably on bloodcharacteristics, e.g. they are hypotriglyceridemic and reduce plateletaggregation. Diacylglycerol reduces serum triacylglycerol levels and canthus have a favourable effect on the risk of cardiovascular disease.Both n-3 fatty acids and diacylglycerols may lower risk ofatherosclerosis and CVD mortality also by other mechanisms.

Other non-limiting examples of the beneficial nutritional componentsinclude dietary fibre and beneficial minor components, for example suchas isoflavones, tocopherols, tocotrienols, carotenoids, vitamin C,folate and flavonoids. Also other vitamins and minerals may be added orincluded in the food products of the present invention.

Especially the use of dietary fibre may further enhance the effects ofthe present compositions. By dietary fibre, it is meant food componentsderived from plant material, or analogous carbohydrates from othersources, that are resistant to digestion and absorption by humandigestive enzymes. This includes various polysaccharides,oligosaccharides, lignins and associated substances that are resistantto digestion. Dietary fibre may further be classified into water-solubleand water insoluble fractions. Examples of the water-soluble fractionare e.g. pectin, plant gums, β-glucans and resistant starch and of theinsoluble fraction e.g. cellulose and hemicellulose. An adequate fibreintake is thought to have many beneficial effects, for example viscoussoluble fibre is known to reduce risk of cardiovascular disease bylowering plasma cholesterol levels. In a further aspect of the presentinvention there is provided a pharmaceutical or nutraceutical productfor improving serum lipid profile comprising a composition of thepresent invention as defined above. Said product can additionallycontain at least one compounding agent. The compounding agent can be anypharmaceutically acceptable binder, carrier, diluent, excipient orcoating agent. The product can be in any suitable form, e.g. tablets,granules, powder, capsules, syrups, dispersions or other liquidpreparations for oral administration.

The products and compositions of the present invention preferablycontain at least two active ingredients, i.e. protein hydrolysate(s) andplant sterol(s), in sufficient amounts to provide improvement in serumlipid profile, e.g. reduction in serum total and/or LDL cholesterollevels. The invention is especially directed to synergistic combinationof at least two active ingredients, i.e. protein hydrolysate(s) andplant sterol(s), the use of which leads to enhanced improvements inserum lipid profile compared to the effects obtained when one of theingredients is used alone. Certain protein hydrolysate/plantsterol/emulsifier complexes according to the present invention areespecially beneficial as the lipid profile improving effect is optimizedby ensuring optimal availability of the active ingredients at thecritical sites of action. In addition to reduction of serum total andLDL cholesterol levels, some compositions of the present invention alsoprovide desired increase in serum HDL cholesterol levels. Some of thecompositions also have a beneficial effect on blood pressure. Theinvention is also directed to combinations of active ingredient(s) andingredient(s) ensuring the optimal availability of the active ingredientat the site of action, i.e. protein hydrolysate(s) and syntheticemulsifier(s).

The present invention also makes it possible to reduce the daily intakeof plant sterols in man and still achieving similar cholesterol loweringeffects as obtained with the generally recommended daily intake of plantsterols (2-3 g) which can be achieved e.g. by consuming commercialproducts enriched with plant sterols. The optimal daily intake of thecompositions of the present invention as such or when used in foods,nutraceuticals or pharmaceuticals is such that a daily intake of plantsterols (calculated as sterol equivalents) of 0.4-5 g, preferably0.5-2.5 g, more preferably 0.8-2 g is supplied.

According to the present invention it is also possible to lower theprotein hydrolysate content of food products containing proteinhydrolysates especially purposed for improving serum lipid profile, thusimproving the organoleptic properties of such foods. The optimal dailyintake of the compositions of the present invention as such or when usedin foods, nutraceuticals or pharmaceuticals is such that a daily intakeof protein hydrolysate of 0.1-60 g, preferably 0.5-15 g, more preferably0.8-10 g is supplied.

The invention is also directed to food products containing at least 10%of the optimal daily intake of the plant sterols and proteinhydrolysates per serving. Preferably the food products contain from 10%to 200% of the optimal daily intake of plant sterols and proteinhydrolysates per serving.

Typically the plant sterol content of the food products is between0.05-20 g per 100 g food product, preferably 0.1-15 g/100 g, morepreferably 0.1-10 g/100 g, and most preferably 0.5-5 g/100 g and theprotein hydrolysate content is between 0.0002-20 g per 100 g foodproduct, preferably 0.0005-15 g/100 g, more preferably 0.001-10 g/100 g,and most preferably 0.1-10 g/100 g.

In yet a further aspect of the present invention there is provided amethod for improving serum lipid profile e.g. for lowering total and/orLDL cholesterol, increasing HDL cholesterol, reducing triacylglycerol,reducing apolipoprotein B and/or increasing the ratio of HDL cholesterolto LDL cholesterol in a subject, comprising orally administering to thesubject a composition according to the present invention in an amounteffective for improving serum lipid profile.

Further, the present invention provides a method to reduce or preventthe development of atherosclerosis in humans by dietary means includingorally administering to the subject a composition according to thepresent invention in an amount effective for improving serum lipidprofile.

The main advantages of the present invention obtained by combining theintake of the two active ingredients are:

-   -   synergistic serum lipid profile improving effect of plant        sterols and protein hydrolysates    -   enhanced serum lipid profile improving effect compared to what        is achievable by using plant sterols or protein hydrolysates        alone    -   lower daily intake of plant sterols is needed to achieve the        same serum lipid profile improving effect than what is achieved        by using plant sterols alone    -   lower daily intake of protein hydrolysates is needed to achieve        the same serum lipid profile improving effect than what is        achieved by using protein hydrolysates alone    -   lowering the risk of cardiovascular disease by means of        improving serum lipid profile.

The present invention is especially directed to enhanced serum lipidprofile improving effect by utilizing the synergistic combination ofplant sterols and protein hydrolysates, optionally with emulsifiersand/or fat and/or mineral salt. The present invention is also directedto ensuring optimal availability of the active ingredients, especiallyprotein hydrolysates, at the site of action. Certain compositionscontaining protein hydrolysates and emulsifiers or protein hydrolysates,emulsifiers and plant sterols according to the current invention areespecially beneficial as the lipid profile improving effect is optimizedby ensuring optimal availability of the active ingredients.

The present invention is also especially directed to an optimized dailyintake of plant sterol and protein hydrolysate to achieve a significantserum lipid profile improving effect in a form of a food product as partof the daily diet. The daily intake of plant sterols can be decreased bycombining the beneficial effect of plant sterols and other lipid profileimproving dietary components to achieve similar lipid profile improvingeffect as obtained with currently recommended daily intake (2-3 g/d) ofplant sterols. This approach advantageously reduces the daily intake ofplant sterols, leading to a more balanced use of plant sterols. Thesynergistic effect is especially profound at daily intakes of at most 2g plant sterol.

In a preferred embodiment of the invention the compositions containplant sterols and soy protein hydrolysate, optionally withemulsifier(s). Preferably the plant sterol used is a plant sterol fattyacid ester and even more preferred it is a plant sterol fatty acid estercontaining a substantial amount of stanol fatty acid ester, preferablyat least 30% stanol fatty acid ester.

The compositions according to the present invention are most preferablyincorporated into foods designed for being a part of a healthy diet.

Additional Aspects of the Invention are:

Use of a composition according to any one of claims 1 to 13 for thepreparation of a pharmaceutical, nutraceutical or food product forimproving serum lipid profile, especially for lowering serum totaland/or LDL cholesterol and/or for increasing the ratio of HDLcholesterol to LDL cholesterol and/or for lowering the serumapolipoprotein B level.

Use of a composition according to any one of claims 1 to 13 for thepreparation of a pharmaceutical, nutraceutical or food product forreducing or preventing the development of atherosclerosis.

The above uses wherein the protein hydrolysate is administrated at arate of 0.1 to 60 g, preferably 0.5 to 15 g and more preferably 0.8 to10 g per day, and the plant sterol is administrated at a rate of 0.4 to5 g, preferably 0.5 to 2.5 g and more preferably 0.8 to 2 g per daycalculated as sterol equivalents.

A method for improving serum lipid profile, especially for loweringserum total and/or LDL cholesterol and/or for increasing the ratio ofHDL cholesterol to LDL cholesterol and/or for lowering the serumapolipoprotein B level in a subject, comprising orally administering tothe subject a composition according to any one of claims 1 to 13, in anamount effective for improving serum lipid profile.

A method for reducing or preventing the development of atherosclerosisin a subject by dietary means including orally administering to thesubject a composition according to any one of claims 1 to 13, in anamount effective for improving serum lipid profile.

The above methods wherein the protein hydrolysate is administrated at arate of 0.1 to 60 g, preferably 0.5 to 15 g and more preferably 0.8 to10 g per day, and the plant sterol is administrated at a rate of 0.4 to5 g, preferably 0.5 to 2.5 g and more preferably 0.8 to 2 g per daycalculated as sterol equivalents.

The invention is further illustrated by the following examples. Examples1-4 illustrate preparation of the constituents of the compositions,examples 5-8 illustrate preparation of the compositions according to theinvention, examples 9-16 illustrate the use of the compositions in foodproducts and example 17 illustrates the effect of a composition of thepresent invention in lowering serum cholesterol. In this specificationthe percentages mean % by weight unless otherwise specified.

EXAMPLE 1

Preparation of Protein Hydrolysate.

1500 g isolated soy protein (SUPRO® Brand Isolated Soy Protein, ProteinTechnologies International) was dispersed in 15 l of water, pH wasadjusted to 2 with HCl, and pepsin (P7000, Sigma Aldrich, activity1:10,000) was added to the solution (1% of the amount of isolated soyprotein). The reaction was carried out at 37° C., for 24 hours andstopped by heating the reaction mixture to 85° C. for one hour. Themixture was neutralized with sodium hydroxide (2 mol/l) and centrifuged.The precipitate was washed twice with water after which the proteinhydrolysate was freeze-dried and ground. 300 g dried protein hydrolysatewas obtained. The molecular weight of the peptides obtained in thehydrolysis was determined by SDS-polyacrylamide gel electrophoresis(SDS-page) method. The molecular weight range of the peptides in thehydrolysate was from 3000 to 30000 D.

EXAMPLE 2

Preparation of Protein Hydrolysate/Emulsifier Complex.

62 g of emulsifier (lysolecithin, Precept 8160, Central Soya) wasdispersed in water (2500 ml) in room temperature and 500 g soy proteinhydrolysate prepared as in example 1 was added. The solution was mixedby a high speed mixer (10 000 rpm), the mixture was freeze-dried andground to obtain protein hydrolysate/emulsifier complex.

The total lipid content of the complex was determined by an acidhydrolysis method (AOAC 922.06). The lipids that were not bound or onlyloosely bound to the complex were determined by direct petroleum etherextraction (free lipids). The content of bound lipids was calculated asthe difference of total and “free” lipids. Total lipid content was 11.0%of the complex, free lipid content 7.9% of the complex and thus the“bound” lipid content was 3.1% of the complex.

EXAMPLE 3

Preparation of Plant Sterol/Emulsifier Complex Using Plant Sterols intheir Free Form.

Plant sterol/emulsifier complex was prepared of plant sterols in theirfree form (Phytosterols, Archer Daniels Midland Company), sodiumstearoyl lactylate (Grindsted SSL P 55, Danisco Cultor) and citric acidester of monoglycerides (Grindsted™ Citrem P 70, Danisco Cultor). Sodiumstearoyl lactylate (30 g, beads) and citric acid esters ofmonoglycerides (70 g, paste) were melted at 60° C. and mixed homogenous.Plant sterols (100 g) were added into the emulsifier mixture, theingredients were mixed together and heated under agitation to about 145°C. Clear liquid obtained was then cooled with stirring to 70° C. andused for preparation of a salad dressing (in the example 11).

EXAMPLE 4

Preparation of Plant Sterol/Emulsifier Complex Using Plant Sterols Bothin their Free and Esterified Forms.

Plant sterol/emulsifier complex was prepared of a mixture of plantsterols in their free and esterified forms and lecithin. Plant sterolester (150 g, Sterol ester-115, Raisio Benecol) was molten at 80° C.Plant sterols (100 g, Phytosterols, Archer Daniels Midland Company),with reduced particle size, and lecithin (100 g, Adlec, Archer DanielsMidland Company) were mixed into molten sterol ester and the mixture washeated under agitation to melt the crystalline plant sterols. Theproduct was homogenized with a high speed mixer (25 000 rpm) and cooledwith stirring to 60° C.

EXAMPLE 5

Preparation of Protein Hydrolysate/Emulsifier Complex by Using aSynthetic Emulsifier.

35 g of emulsifier (citric acid ester of monoglycerides, Grindsted™Citrem P 70, Danisco Cultor) was dispersed in water (1500 ml) at 50° C.and 150 g of the soy protein hydrolysate prepared in example 1 wasadded. The solution was mixed by a high speed mixer (25 000 rpm), themixture was freeze-dried and ground to obtain proteinhydrolysate/emulsifier complex.

EXAMPLE 6

Preparation of Protein Hydrolysate/Plant Sterol Complex Using PlantSterols in their Free Form.

Isolated soy protein (500 g, SUPRO® Brand Isolated Soy Protein, ProteinTechnologies International) was hydrolyzed as in example 1 and washedtwice with water after hydrolyzation. Plant sterols in their free form(40 g, Phytosterols, Archer Daniels Midland Company) were added andhomogenized with the washed protein hydrolysate with a high speed mixer(25 000 rpm). The composition was freeze-dried and ground.

EXAMPLE 7

Preparation of Protein Hydrolysate/Plant Sterol Complex ContainingMineral Salt, Using Plant Sterols in their Esterified Form.

Isolated soy protein (500 g, SUPRO® Brand Isolated Soy Protein, ProteinTechnologies International) was hydrolyzed as in example 1 and washedtwice with water after hydrolyzation. Mineral salt (5 g, Pansalt®) wasadded to the washed hydrolysate slurry and the slurry was freeze-driedand ground. 34.4 g plant sterol ester (Sterol ester-115, Raisio Benecol)was added to 65.6 g of the dried hydrolysate. The mixture washomogenized with a high speed mixer (25 000 rpm).

EXAMPLE 8

Preparation of Protein Hydrolysate/Plant Sterol/Emulsifier Complex.

Soy protein hydrolysate was prepared as in example 1. Enzyme modifiedlecithin (20 g, Precept 8160, Central Soy Company) and citric acidesters of monoglycerides (50 g Grindsted™ Citrem P 70, Danisco Cultor)were melted at 60° C. and mixed homogenous. Plant sterols (50 g,Phytosterols, Archer Daniels Midland Company) with reduced particle sizewere added into the emulsifier mixture, the ingredients were mixedtogether and heated under agitation to 100° C. Clear liquid obtained wasthen cooled with stirring until the temperature reached 90° C. Dried andground soy protein hydrolysate (90 g) was added and homogenized into theplant sterol emulsifier blend at 90° C. The mixture was cooled to 70° C.and used for preparation of a milk alternative (in the example 15) and afruit drink (in the example 16).

The weight ratio of plant sterol to protein hydrolysate in the complexwas 1:1.8 and the complex contained 23.8% plant sterols and 42.9% soyprotein hydrolysate.

EXAMPLE 9

Food Bar with Protein Hydrolysate/Plant Sterol Complex.

Food Bar was Prepared of the Following Ingredients:  300 g oat flakes 95 g fiber rich oat bran  140 g corn syrup  50 g brown sugar  40 gconcentrated apple juice  20 g apple  30 g raisin  100 g vegetable fat  3 g salt   8 g flavoring  110 g complex from example 6   4 g lecithinA 45 g food bar contained 1.6 g plant sterols and 3.9 g soy proteinhydrolysate.

EXAMPLE 10

Yogurt with Protein Hydrolysate/Emulsifier Complex and Plant Sterols.

Plant stanol ester (STAEST-115, Raisio Benecol) and proteinhydrolysate/emulsifier complex from example 5 were used separately asingredients in preparation of yogurt. Plant stanol ester (STAEST-115)was molten at 70° C. Molten stanol ester (110 g) and 140 g soy proteinhydrolysate/emulsifier complex from example 5 were added into 5,75 l ofskimmed milk at 70° C., mixed with a high speed mixer and the mixturewas pasteurized. Normal yogurt cultures and Bifidobacteria were stirredinto the mixture and the mixture was held at 42° C. for 7 hours.

A 150 g serving of the product contained 1.7 g plant stanols and 3.0 gprotein hydrolysate.

EXAMPLE 11

Low-Fat Salad Dressing with Plant Sterol/Emulsifier Complex and ProteinHydrolysate.

Plant sterol/emulsifier complex from example 3 and soy proteinhydrolysate from example 1 were used separately as ingredients in apreparation of low-fat salad dressing.

Ingredients of the Salad Dressing were: 7.5 g plant sterol/emulsifiercomplex from example 3   7 g soy protein hydrolysate from example 1  16g soybean oil  56 g water   6 g vinegar 4.5 g sugar 1.5 g salt 0.1 gxanthan gum 0.4 g lemon juice   1 g spices  30 g serving of the saladdressing contained 1.1 g plant sterols and 2.1 g soy proteinhydrolysate.

EXAMPLE 12

Yogurt-Like Cereal Product with Plant Sterol/Emulsifier Complex andProtein Hydrolysate.

Protein hydrolysate was prepared from wheat gluten (Raisio) in a similarway as soy protein hydrolysate in example 1, except the hydrolysate wasnot dried. Water was added into the washed gluten hydrolysate to obtaina product having a solid content of 20%.

Plant sterol/emulsifier complex from example 4 and wheat glutenhydrolysate were used in a preparation of fermented, yogurt-like cerealproduct.

Ingredients   45% water 16.5% prepared mixture of gluten hydrolysate andwater  8.9% oat bran   28% berry jam (containing fructose, blueberry,strawberry, raspberry, pectin, flavors)  1.6% plant sterol/emulsifiercomplex from example 4

The mixture of water and oat bran was fermented using Bifidobacteriaculture. The berry jam, sterol/emulsifier complex and mixture of glutenhydrolysate and water were added and all ingredients worked together.

A 150 g serving of the product contained 1.3 g plant sterols and 5.0 gprotein hydrolysate.

EXAMPLE 13

Bread Rolls with Protein Hydrolysate/Plant Sterol Complex ContainingMineral Salt.

Bread Rolls Containing the Complex from Example 7 were Prepared in aConventional Way of the Following Ingredients: 800 g wheat flour  20 gsugar  20 g salt  10 g margarine (80% fat content)  94 g complex fromexample 7 500 g water  11 g dried yeast  24 bread rolls were obtainedfrom the dough. Two 50 g bread rolls contained suitable daily doses ofplant sterols (1.3 g) and protein hydrolysate (4.0 g).

EXAMPLE 14

Cream Cheese Style Spread with Plant Sterols and Protein Hydrolysatewith Mineral Salt.

Plant stanol ester (STAEST-115, Raisio Benecol) and soy proteinhydrolysate with mineral salt were used separately as ingredients in apreparation of cream cheese style spread. Soy protein hydrolysatecontaining mineral salt was prepared in a similar way as the complex inexample 7, except no plant sterol ester was added into the dried andground protein hydrolysate. The dried soy protein hydrolysate contained4.8% mineral salt.

Plant stanol ester was first incorporated into a fat blend, compositionof which was 59.7% rapeseed oil, 7% interesterified blend of palmstearine and coconut oil and 33.3% plant stanol ester (STAEST-115,Raisio Benecol). The blend was prepared by blending the melted stanolester with rapeseed oil and the hardstock component.

The Cream Cheese Style Spread was Produced According to the FollowingRecipe: 51.3% curd 24.6% fat blend including the stanol ester 12.0%condensate  1.0% stabilizer  1.0% milk proteins  7.9% soy proteinhydrolysate containing 4.8% mineral salt  0.3% salt  0.1% potassiumsorbate  1.7% garlic flavor preparation 0.05% lactic acid aspH-regulating agent flavors

A serving of 20 g of the cream cheese style spread contained 1 g plantsterols (as plant stanols) and 1.5 g protein hydrolysate (as soy proteinhydrolysate).

EXAMPLE 15

Milk Alternative with Protein Hydrolysate/Plant Sterol/EmulsifierComplex.

Protein hydrolysate/plant sterol/emulsifier complex from example 8 wasused in production of vanilla flavored milk alternative.

Ingredients   5 l soymilk (2% fat) 94.5 g complex from example 8 vanillaflavoring

The soy protein hydrolysate/plant sterol/emulsifier complex from example8 was added under vigorous stirring to soymilk having a temperature of70° C. and homogenized. 2 dl serving of the soymilk contained 0.9 g ofplant sterols and 1.6 g of soy protein hydrolysate.

EXAMPLE 16

Fruit Drink with Protein Hydrolysate/Plant Sterol/Emulsifier Complex.

The Protein Hydrolysate/Plant Sterol/Emulsifier Complex from Example 8was Used in Preparation of Fruit Drink of the Following Ingredients: 200 g fruit juice concentrate (orange, pineapple, passion fruit, guava,mango)  776 g water  8.5 g fructose  1.5 g calcium lactate   14 gcomplex from example 8

The fruit juice concentrate, water, fructose and calcium lactate weremixed together and heated to 70° C. The soy protein hydrolysate/plantsterol/emulsifier complex from example 8 was added under vigorousstirring to the drink and homogenized.

Two glasses (á 2 dl) of the drink contained 1.3 g of plant sterols and2.4 g of protein hydrolysate.

EXAMPLE 17

Lipid Profile Improvement Obtained by Using a Composition of the PresentInvention.

The lipid profile improving effect of a composition containing plantsterols (as plant stanol fatty acid ester) and proteinhydrolysate/emulsifier complex (as soy protein hydrolysate/lysolecithincomplex from example 2) was studied by using LDL-receptor deficientfemale mice as test animals.

The aim of the test was to study the serum total cholesterol andtriglyceride lowering effect that could be obtained by using only asmall amount of plant sterols (0.5%, as sterol equivalents) as part of aatherogenic diet containing protein hydrolysate. The protein hydrolysatewas administered in an amount to yield the maximum cholesterol loweringeffect that could be obtained by using the hydrolysate in this animalmodel and still not compromising the nutritional needs and growth of themice. The suitable amount of the hydrolysate was found to be ½ (as Nequivalents) of the total protein content of the diet.

The animals were assigned into 4 groups (n=8-10 in each group) and fedexperimental diets for 8 weeks.

All the experimental diets were formulated to contain 20% protein. Inthe control group and in the test group 2, the sole protein source wascasein (88% purity). In the test groups 3 and 4, the protein source washalf casein and half (as N equivalents) the proteinhydrolysate/emulsifier complex. Test groups 2 and 4 contained 0.84%plant stanol fatty acid ester (0.5% as sterol equivalents). Controlgroup and test group 3 contained 0.35% rapeseed oil to bring the equalamount of calories and equal fatty acid composition, compared to thetest groups getting plant stanol ester.

Experimetal Diets and Results: Group 3 (protein Group 4 Group 2hydrolysate/ (plant stanol ester + Group 1 (plant stanol emulsifierprotein hydrolysate/ (control) ester) complex) emulsifier complex)Ingredients % of total % of total % of total % of total Diet premix*58.72 58.22 55.44 54.95 Cocoa butter 17.96 17.96 17.97 17.97 Rapeseedoil 0.35 0.00 0.35 0.00 Plant stanol ester 0.00 0.84 0.00 0.84Caseinpowder 22.72 22.73 11.36 11.37 Soy protein hydrolysate/ 0.00 0.0014.62 14.62 lysolecithin complex, from example 2 Cholesterol 0.250 0.2500.250 0.250 Results Serum total cholesterol 20.6 14.7 15.5 11.4(mmol/l), mean Change (%) compared to — −28.5 −25.0 −44.5 control groupSerum triglycerides 2.2 2.4 1.9 1.3 (mmol/l), mean Change (%) comparedto +8.8 −10.9 −39.1 control group*Clinton/Cybulsky (D12106px, without protein), from Research Diets Inc.

As shown in the table, both plant stanol ester and proteinhydrolysate/emulsifier complex had serum cholesterol lowering effect(groups 2 and 3, respectively). A composition of the present invention(group 4), the combination of plant sterols and proteinhydrolysate/emulsifier complex, had even enhanced cholesterol loweringeffect compared to the plant sterols (group 2) or proteinhydrolysate/emulsifier complex (group 3) alone and compared to what wasexpected for the combination. Thus even when the maximal amount ofprotein hydrolysate was used in the feed addition of plant sterolseffectively further reduced serum total cholesterol levels

Protein hydrolysate/emulsifier complex (group 3) had serum triglyceridereducing effect, whereas the serum triglyceride level was somewhatraised in the group 2 receiving plant sterols. Surprisingly, thecombination of plant sterols and protein hydrolysate/emulsifier complexhad a strong synergistic triglyceride lowering effect.

By combining plant sterols and protein hydrolysate, according to thepresent invention, remarkable synergistic lipid profile improvementscould be seen.

1-28. (canceled)
 29. A therapeutical composition comprising a proteinhydrolysate and a plant sterol, wherein the weight ratio of the plantsterol to the protein hydrolysate is from 1:0.02 to 1:150.
 30. Thecomposition according to claim 29, comprising an additional componentselected from the group consisting of emulsifiers, fats, mineral saltsand mixtures thereof.
 31. The composition according to claim 29, whereinthe plant sterol is selected from the group consisting of free sterols,free stanols, sterols in esterified form, stanols in esterified form andmixtures thereof.
 32. The composition according to claim 29, wherein theprotein hydrolysate is derived from vegetable or animal origin,including soybean protein, wheat protein, corn protein, oat protein, ryeprotein, rapeseed protein, barley protein, milk protein and mixturesthereof.
 33. The composition according to claim 30, wherein theemulsifier is selected from the group consisting of monoglycerides,diglycerides, monoglyceride derivatives, lecithins, modified lecithins,polyglycerol esters, polyglycerol polyricinoleate, sorbitan esters,polysorbates, propylene glycol esters, stearoyl lactylates, diacetyltartaric acid esters, diacetyl lactic acid esters, sucrose esters andmixtures thereof.
 34. The composition according to claim 30, wherein theemulsifier has a hydrophilic lipophilic balance (HLB) of at least 4,preferably at least
 6. 35. The composition according to claim 29,wherein the weight ratio of the plant sterol to the protein hydrolysateis from 1:0.2 to 1:30, preferably from 1:0.4 to 1:12.5 and morepreferably from 1:1 to 1:5.
 36. The composition according to claim 30,wherein the protein hydrolysate is in the form of a proteinhydrolysate/emulsifier complex.
 37. The composition according to claim36, wherein the complex comprises at least 5%, preferably at least 10%,more preferably at least 20% emulsifier on dry weight basis.
 38. Thecomposition according to claim 30, wherein the plant sterol is in theform of a plant sterol/emulsifier complex or dissolved or suspended infat or a mixture of fat and an emulsifier.
 39. The composition accordingto claim 30, wherein the protein hydrolysate and the plant sterol are inthe form of a protein hydrolysate/plant sterol/emulsifier complex. 40.The composition according to claim 38, wherein the weight ratio of theplant sterol to emulsifier is from 1:0.01 to 1:5, preferably from 1:0.05to 1:2, more preferably from 1:0.1 to 1:2.
 41. The composition accordingto claim 30, wherein the fat content is from 0 to 80%, preferably from 0to 60%, more preferably from 0 to 20% of the weight of the complex orsuspension or solution.
 42. A therapeutical composition comprising aprotein hydrolysate and at least one emulsifier or a mixture of at leastone emulsifier and a fat, wherein the emulsifier is a syntheticemulsifier.
 43. A therapeutical composition comprising a proteinhydrolysate and at least one emulsifier or a mixture of at least oneemulsifier and a fat, wherein the emulsifier is a lipid based grainfraction having emulsifying properties.
 44. A food product comprising atleast one basic nutritional ingredient and a composition according toclaim
 29. 45. The food product according to claim 44 being in the formof a product selected from the group consisting of bakery,confectionery, cereal, fermented cereal, snacks, beverage, dairy, sauce,soup, meat, fish, poultry, egg, soy based, vegetable oil based and readymix products.
 46. The food product according to claim 44, wherein theplant sterol content calculated as sterol equivalents is from 0.05 to 20g per 100 g food product and the protein hydrolysate content is from0.0002 to 20 g per 100 g food product.
 47. A pharmaceutical ornutraceutical product comprising a composition according to claim 29.48. A composition according to claim 29 for use as a pharmaceutical ornutraceutical product.
 49. A composition according to claim 29 for usein a pharmaceutical, nutraceutical or food product for improving serumlipid profile, especially for lowering serum total and/or LDLcholesterol and/or for increasing the ratio of HDL cholesterol to LDLcholesterol and/or for lowering the serum apolipoprotein B level and/orfor lowering the serum triglyceride level, and/or for lowering bloodpressure.
 50. The food product, pharmaceutical or nutraceutical productor composition according to claim 44, wherein the amounts of the proteinhydrolysate and the plant sterol are such that the protein hydrolysateis consumed at a rate of 0.1 to 60 g, preferably 0.5 to 15 g and morepreferably 0.8 to 10 g per day, and the plant sterol is consumed at arate of 0.4 to 5 g, preferably 0.5 to 2.5 g and more preferably 0.8 to 2g per day calculated as sterol equivalents.
 51. Use of a compositionaccording to claim 29 for the preparation of a pharmaceutical,nutraceutical or food product for improving serum lipid profile,especially for lowering serum total and/or LDL cholesterol and/or forincreasing the ratio of HDL cholesterol to LDL cholesterol and/or forlowering the serum apolipoprotein B level and/or for lowering the serumtriglyceride level, and/or for lowering blood pressure.
 52. Use of acomposition according to claim 29 for the preparation of apharmaceutical, nutraceutical or food product for reducing or preventingthe development of atherosclerosis.
 53. The use according to claim 51,wherein the protein hydrolysate is administrated at a rate of 0.1 to 60g, preferably 0.5 to 15 g and more preferably 0.8 to 10 g per day, andthe plant sterol is administrated at a rate of 0.4 to 5 g, preferably0.5 to 2.5 g and more preferably 0.8 to 2 g per day calculated as sterolequivalents.
 54. A method for improving serum lipid profile, especiallyfor lowering serum total and/or LDL cholesterol and/or for increasingthe ratio of HDL cholesterol to LDL cholesterol and/or for lowering theserum apolipoprotein B level and/or for lowering the serum triglyceridelevel in a subject, and/or for lowering blood pressure in a subject,comprising orally administering to the subject a composition accordingto claim 29, in an amount effective for improving serum lipid profileand/or for lowering blood pressure.
 55. A method for reducing orpreventing the development of atherosclerosis in a subject by dietarymeans including orally administering to the subject a compositionaccording to claim 29, in an amount effective for improving serum lipidprofile.
 56. The method according to claim 54, wherein the proteinhydrolysate is administrated at a rate of 0.1 to 60 g, preferably 0.5 to15 g and more preferably 0.8 to 10 g per day, and the plant sterol isadministrated at a rate of 0.4 to 5 g, preferably 0.5 to 2.5 g and morepreferably 0.8 to 2 g per day calculated as sterol equivalents.